1. Field of the Invention
The present invention relates to gene insertion in Zymomonas mobilis, and in particular to the insertion inactivation of specific gene products in recombinant Zymomonas mobilis strains which ferment xylose and arabinose, or both, into ethanol.
2. Description of the Related Art
Fermentation technology is useful for the conversion renewable biomass cellulose substrates into fuels and chemicals, such as ethanol. A typical substrate is comprised of 35-45% cellulose, 30-40% hemicellulose, and 15% lignin. The hydrolysis fraction contains glucose polymers, and the hemicellulose fraction contains mostly xylose. Arabinose is also a significant fermentable substrate found in biomass materials, such as switchgrass grass and corn fiber.
Z. mobilis is widely reported for its ability to rapidly and efficiently convert glucose substrates into ethanol, at a low pH, in an anaerobic culture, and in a medium which contains the inhibitory compounds typically associated with a lignocellulose-hydrolysate. A distinct disadvantage in the use of Z. mobilis is, however, that it does not ferment pentose sugars. To overcome this disadvantage, the prior art has focused on recombinant Z. mobilis strains which ferment a mixture of glucose, and xylose or arabinose, or both, using exogenous genes which catalyze the metabolism of xylose and arabinose. These strains are based on the use of multicopy plasmids capable of expression of the desired enzyme product.
U.S. Pat. No. 5,514,583 discloses a transformed Z. mobilis xylose fermenting strain (CP4/pZB4 and pZB5) having exogenous genes, and plasmid vectors (pZB4 and pZB5) encoding xylose isomerase, xylulokinase, transaldolase and transketolase, and further comprising at least one promoter (Pgap and Peno) recognized by Zymomonas which regulates the expression of at least one of said genes. The microorganism is capable of growing on xylose as a sole carbon source, and fermenting xylose to ethanol at about 88% of the maximum theoretic yield. The patent claims an integrated strain.
U.S. Pat. Nos. 5,712,133 and 5,726,053 disclose, inter alia, Z. mobilis arabinose fermenting transformants (CP4/pZB 206), containing exogenous genes that encode L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4-epimerase, transaldolase and transketolase which impart arabinose to ethanol fermentation capability. The plasmid vector (pZB 206) and a process of using the transformants for the fermentation of a glucose and arabinose containing substrate is also disclosed. The patent claims integration of the exogenous genes into the host genome.
U.S. Pat. No. 5,843,760 discloses a Z. mobilis xylose and arabinose fermenting transformant (206C/pZB301) containing exogenous genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase and transketolase, and further comprising at least one promoter recognized by Zymomonas which regulates the expression of at least one of said genes, wherein said microorganism is capable of growing on arabinose and/or xylose, alone or in combination, as the carbon source and fermenting said arabinose and xylose to ethanol. The process of using the transformants together with the plasmid vectors (pZB301, pZB401, pZB402, and pZB 403) is also disclosed. This patent claims integration of the exogenous genes into the host genome.
Vital to the engineering of an economic fermentation process for the production of fuels and chemicals, from cellulose containing substrates, is to achieve a high rate of specific product formation and conversion efficiency. When using the pentose-fermenting recombinant strains of Z. mobilis, where ethanol is the specific product, the formation of the by-products lactic acid, an end product of energy formation, and xylitol lowers conversion efficiency. Thus, it is desirable to develop new metabolic engineering methods for the site-specific insertion of genes in Z. mobilis, such as for the insertion inactivation of lactate dehydrogenase, to enhance the conversion efficiency in a cellulose to ethanol process.
In Esherichia coli, the classical method for generating chromosomal inserts of foreign genes involves the use of specialized λ phage cloning vectors that can exist stable in the lysogenic state. Alternatively, site specific genes can be inserted though homologous recombination, when bracketed with E. coli chromosomal sequences, or by random transposition if the genes can be cloned in the permissive sites of a transposon. While transposition has been demonstrated in Z. mobilis, Pappas, K. M., et al., (1997) Transposon mutagensesis and strain construction in Zymomonas mobilis, Journal of Applied Microbiology, Vol. 82, p.p. 379-388 (Tn5 or mini Mμ transposition of auxotrophy or antibiotic resistance for genetic analysis in Z. mobilis) transposition is random and homologous recombination in Z. mobilis has not been demonstrated. Further, no bacteriophage has ever been isolated from Zymomonas. 
In view of the foregoing, a need exists for a method of site specific insertion in Z. mobilis. One practical application of the method would be for the elimination in the formation of by-products, in a Z. mobilis fermentation, through the construction of stable recombinant strains, characterized by the insertion inactivation of genes encoding those enzymes in the specific by-product formation pathway, to be eliminated.